Genetic suppression and replacement

ABSTRACT

A strategy for suppressing specifically or partially specifically an endogenous gene and introducing a replacement gene, said strategy comprising the steps of: 1. providing suppressing nucleic acids or other suppression effectors able to bind to an endogenous gene, gene transcript or gene product to be suppressed and 2. providing genomic DNA or cDNA (complete or partial) encoding a replacement gene wherein the suppressing nucleic acids are unable to bind to equivalent regions in the genomic DNA or cDNA to prevent expression of the replacement gene. The replacement nucleic acids have modifications in one or more third base (wobble) positions such that replacement nucleic acids still code for the wild type or equivalent amino acids.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.09/155,708, which was filed under 35 U.S.C. §371 for, and claimspriority to, PCT/GB97/00929, filed Apr. 2, 1997, which claims priorityto GB9606961.2, filed Apr. 2, 1996, the disclosures of which areincorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to a strategy for suppressing a gene. Inparticular the invention relates to suppression of a mutated gene thatgives rise to a dominant or deleterious effect, either monogenically orpolygenically.

BACKGROUND OF THE INVENTION

Studies of degenerative hereditary ocular conditions, includingRetinitis Pigmentosa (RP) and various macular dystrophies, have resultedin a substantial elucidation of the molecular basis of thesedebilitating human retinal degenerations. Applying the approach ofgenetic linkage, x-linked RP (xlRP) genes have been localised to theshort arm of the X chromosome (Ott et al. 1990). Subsequently, the geneinvolved in one form of xlRP was identified. Various genes involved inautosomal dominant forms of RP (adRP) have been localised. The first ofthese mapped to 3q, close to the gene encoding the rod photoreceptorprotein rhodopsin (McWilliam et al. 1989; Dryja et al. 1990). Similarly,an adRP gene was placed on 6p close to the gene encoding thephotoreceptor protein peripherin (Farrar et al. 1991a,b; Kajiwara et al.1991). Other adRP genes have been mapped to discrete chromosomallocations; however the disease genes as yet remain uncharacterised. Asin xlRP and adRP, various genes involved in autosomal recessive RP(arRP) have been localised and in some cases molecular defectscharacterised (Humphries et al. 1992; Farrar et al. 1993; Van Soest etal. 1994). Similarly, a number of genes involved in macular dystrophieshave been mapped (Mansergh et al. 1995). Genetic linkage, together withtechniques for mutational screening of candidate genes, enabledidentification of causative dominant mutations in the genes encodingrhodopsin and peripherin. Globally, about 100 rhodopsin mutations havebeen found in patients with RP or congenital stationary night blindness.Similarly, approximately 40 mutations have been characterised in theperipherin gene in patients with RP or macular dystrophies. Knowledge ofthe molecular aetiology of these retinopathies has stimulated thegeneration of animal models and the exploration of methods oftherapeutic intervention (Farrar et al. 1995; Humphries et al. 1997).

Similar to RP, osteogenesis imperfecta (OI) is an autosomal dominantlyinherited human disorder whose molecular pathogenesis is extremelygenetically heterogeneous. OI is often referred to as ‘brittle bonedisease’, although additional symptoms including hearing loss, growthdeficiency, bruising, loose joints, blue sclerae and dentinogenesisimperfecta are frequently observed (McKusick, 1972). Mutations in thegenes encoding the two type I collagen chains (collagen 1A1 and 1A2)comprising the type I collagen heterodimer have been implicated in OI.Indeed hundreds of dominantly acting mutations have been identified inOI patients in these two genes, many of which are single pointmutations, although a number of insertion and deletion mutations havebeen found (Willing et al. 1993; Zhuang et al. 1996). Similarlymutations in these genes have also been implicated in Ehlers-Danlos andMarfan syndromes (Dalgleish et al. 1986; Phillips et al. 1990; D'Alessioet al. 1991; Vasan NS et al. 1991).

Generally, gene therapies utilising viral and non-viral delivery systemshave been used to treat inherited disorders, cancers and infectiousdiseases. However, many studies have focused on recessively inheriteddisorders, the rationale being that introduction and expression of thewild type gene may be sufficient to prevent/ameliorate the diseasephenotype. In contrast gene therapy for dominant disorders requiressuppression of the dominant disease allele. Notably many of thecharacterised mutations causing inherited diseases such as RP or OI areinherited in an autosomal dominant fashion. Indeed there are over 1,000autosomal dominantly inherited disorders in man. In addition, there aremany polygenic disorders due to co-inheritance of a number of geneticcomponents which together give rise to the disease state. Effective genetherapies for dominant or polygenic diseases may be targeted to theprimary defect and in this case may require suppression of the diseaseallele while in many cases still maintaining the function of the normalallele. This is particularly relevant where disease pathology is due toa gain of function mutation rather than due to reduced levels of wildtype protein. Alternatively, suppression therapies may be targeted tosecondary effects associated with the disease pathology, for example,programmed cell death (apoptosis), which has been observed in manyinherited disorders.

Strategies to differentiate between normal and disease alleles and toselectively switch off the disease allele using suppression effectorssuch as antisense DNA/RNA, PNAs, ribozymes, or triple helix forming DNA,targeted towards the disease mutation may be difficult in manycases—frequently disease and normal alleles differ by only a singlenucleotide. A further difficulty inhibiting development of genetherapies is the heterogeneous nature of some dominant disorders—manydifferent mutations in the same gene give rise to a similar diseasephenotype. Development of specific gene therapies for each of these maybe prohibitive in terms of cost.

Suppression effectors have been used previously to achieve specificsuppression of gene expression. Antisense DNA and RNA has been used toinhibit gene expression in many instances. Modifications, such asphosphorothioates, have been made to oligonucleotides to increaseresistance to nuclease degradation, binding affinity and uptake(Cazenave et al. 1989; Sun et al. 1989; McKay et al. 1996; Wei et al.1996). In some instances, using antisense and ribozyme suppressionstrategies has led to reversal of a tumor phenotype by reducingexpression of a gene product or by cleaving a mutant transcript at thesite of the mutation (Carter and Lemoine 1993; Lange et al. 1993; Valeraet al. 1994; Dosaka-Akita et al. 1995; Feng et al. 1995; Quattrone etal. 1995; Ohta et al. 1996). For example, neoplastic reversion wasobtained using a ribozyme targeted to an H-ras mutation in bladdercarcinoma cells (Feng et al. 1995). Ribozymes have also been proposed asa means of both inhibiting gene expression of a mutant gene and ofcorrecting the mutant by targeted trans-splicing (Sullenger and Cech1994; Jones et al. 1996). Ribozymes can be designed to elicitautocatalytic cleavage of RNA targets; however, the inhibitory effect ofsome ribozymes may be due in part to an antisense effect due to theantisense sequences flanking the catalytic core that specify the targetsite (Ellis and Rodgers 1993; Jankowsky and Schwenzer 1996). Ribozymeactivity may be augmented by the use of, for example, non-specificnucleic acid binding proteins or facilitator oligonucleotides (Herschlaget al. 1994; Jankowsky and Schwenzer 1996). Multitarget ribozymes(connected or shotgun) have been suggested as a means of improvingefficiency of ribozymes for gene suppression (Ohkawa et al. 1993).Triple helix approaches have also been investigated forsequence-specific gene suppression—triplex forming oligonucleotides havebeen found in some cases to bind in a sequence-specific manner (Postelet al. 1991; Duval-Valentin et al. 1992; Hardenbol and Van Dyke 1996;Porumb et al. 1996). Similarly, peptide nucleic acids have been shown toinhibit gene expression (Hanvey et al. 1992; Knudson and Nielsen 1996;Taylor et al. 1997). Minor groove binding polyamides can bind in asequence-specific manner to DNA targets and hence may represent usefulsmall molecules for future suppression at the DNA level (Trauger et al.1996). In addition, suppression has been achieved by interference at theprotein level using dominant negative mutant peptides and antibodies(Herskowitz 1987; Rimsky et al. 1989; Wright et al. 1989). In some casessuppression strategies have lead to a reduction in RNA levels without aconcomitant reduction in proteins, whereas in others, reductions in RNAhave been mirrored by reductions in protein.

SUMMARY OF THE INVENTION

To circumvent difficulties associated with specifically targeting adisease mutation and with the genetic heterogeneity present in inheriteddisorders, a novel strategy for gene suppression and gene replacementexploiting the degeneracy of the genetic code is described. Theinvention allows flexibility in choice of target sequence forsuppression and provides a means of gene suppression that is independentof the disease mutation.

In summary, the invention involves gene suppression of disease andnormal alleles targeting coding sequences in a gene and, when necessary,gene replacement such that the replacement gene cannot be suppressed.Replacement genes are modified at third base positions (wobblepositions) so that they code for the correct amino acids but areprotected completely or partially from suppression. The same suppressionand replacement steps can be used for many disease mutations in a givengene. Suppression and replacement can be undertaken in conjunction witheach other or separately.

The invention relates to a strategy for suppressing a gene or diseaseallele using methods that do not target the disease allele specificallybut instead can be targeted towards a broad range of sequences in aparticular gene. A particular embodiment of the invention is the use ofsuppression strategies to target either the disease or normal allelesalone or to target both disease and normal alleles. A further embodimentof the invention is the use of the wobble hypothesis to enable continuedexpression of a replacement normal or beneficial gene (a gene modifiedfrom the wild type such that it provides an additional beneficialeffect(s)). The replacement gene will have nucleotide changes from theendogenous wild type gene but will code for identical amino acids as thewild type gene. The strategy circumvents the need for a specific therapyfor every mutation within a given gene. In addition, the inventionallows greater flexibility in choice of target sequence for suppressionof a disease allele.

The invention also relates to a medicament or medicaments for use insuppressing a deleterious allele that is present in a genome of one ormore individuals or animals.

Generally, the present invention will be useful where the gene, which isnaturally present in the genome of a patient, contributes to a diseasestate. Generally, one allele of the gene in question will be mutated,that is, will possess alterations in its nucleotide sequence thataffects the function or level of the gene product. For example, thealteration may result in an altered protein product from the wild typegene or altered control of transcription and processing. Inheritance orsomatic acquisition of such a mutation can give rise to a diseasephenotype or can predispose an individual to a disease phenotype.However the gene of interest could also be of wild type phenotype, butcontribute to a disease state in another way such that the suppressionof the gene would alleviate or improve the disease state or improve theeffectiveness of an administered therapeutic compound.

Generally, suppression effectors such as nucleic acids—antisense orsense, ribozymes, peptide nucleic acids (PNAs), triple helix formingoligonucleotides, peptides and/or antibodies directed to sequences in agene, in transcripts or in protein, can be employed in the invention toachieve gene suppression.

DETAILED DESCRIPTION OF THE INVENTION

The invention addresses shortcomings of the prior art by providing anovel approach to the design of suppression effectors directed to targetalleles of a gene carrying a deleterious mutation. Suppression of everymutation giving rise to a disease phenotype may be costly andproblematic. Disease mutations are often single nucleotide changes. As aresult differentiating between the disease and normal alleles may bedifficult. Some suppression effectors require specific sequence targets,for example, hammerhead ribozymes cleave at NUX sites and hence may notbe able to target many mutations. Notably, the wide spectrum ofmutations observed in many diseases adds additional complexity to thedevelopment of therapeutic strategies for such disorders—some mutationsmay occur only once in a single patient. A further problem associatedwith suppression is the high level of homology present in codingsequences between members of some gene families. This can limit therange of target sites for suppression that will enable specificsuppression of a single member of such a gene family.

The present invention circumvents shortcomings in the prior art byutilising the degeneracy of the genetic code. In the inventionsuppression effectors are designed specifically to sequences in codingregions of genes or in gene products. Typically, one allele of the genecontains a mutation with a deleterious effect. Suppression targeted tocoding sequences provides greater flexibility in choice of targetsequence for suppression in contrast to suppression directed towardssingle disease mutations. Additionally, the invention provides for theintroduction of a replacement gene with modified sequences such that thereplacement gene is protected from suppression. The replacement gene ismodified at third base wobble positions and hence provides the wild typegene product. Notably, the invention has the advantage that the samesuppression strategy could be used to suppress, in principle, manymutations in a gene. This is particularly relevant when large numbers ofmutations within a single gene cause disease pathology. The replacementgene provides (when necessary) expression of the normal protein productwhen required to ameliorate pathology associated with reduced levels ofwild type protein. The same replacement gene could in principle be usedin conjunction with the suppression of many different disease mutationswithin a given gene. Target sequences may be found in any part of thecoding sequence. Suppression in coding sequence holds the advantage thatsuch sequences are present in both precursor and mature RNAs, therebyenabling suppressors to target all forms of RNA.

There is now an armament with which to obtain gene suppression. This, inconjunction with a better understanding of the molecular aetiology ofdisease, results in an ever increasing number of disease targets fortherapies based on suppression. In many cases, complete suppression ofgene expression has been difficult to achieve. Possibly a combinedapproach using a number of suppression effectors may aid in this. Forsome disorders it may be necessary to block expression of a diseaseallele completely to prevent disease symptoms whereas for others lowlevels of mutant protein may be tolerated. In parallel with an increasedknowledge of the molecular defects causing disease has been therealisation that many disorders are genetically heterogeneous. Examplesin which multiple genes and/or multiple mutations within a gene can giverise to a similar disease phenotype include osteogenesis imperfecta,familial hypercholesterolemia, retinitis pigmentosa, and many others.The utility of the degeneracy of the genetic code (wobble hypothesis) toenable suppression of one or both alleles of a gene and the introductionof a replacement gene such that it escapes suppression has beenexploited in the invention.

According to the present invention there is provided a strategy forsuppressing expression of an endogenous gene with a deleteriousmutation, wherein said strategy comprises providing suppressioneffectors such as antisense nucleic acids able to bind to sequences of agene to be suppressed, to prevent the functional expression thereof.

Generally the term suppression effectors means nucleic acids, peptidenucleic acids (PNAs), peptides, antibodies or modified forms of theseused to silence or reduce gene expression in a sequence specific manner.

Suppression effectors, such as antisense nucleic acids can be DNA orRNA, can typically be directed to coding sequence; however suppressioneffectors can be targeted to coding sequence and can also be targeted to5′ and/or 3′ untranslated regions and/or introns and/or control regionsand/or sequences adjacent to a gene or to any combination of suchregions of a gene. Antisense nucleic acids including both coding andnon-coding sequence can be used if required to help to optimisesuppression. Binding of the suppression effector(s) prevents or lowersfunctional expression of the endogenous gene.

Generally the term ‘functional expression’ means the expression of agene product able to function in a manner equivalent to, or better than,a wild type product. In the case of a mutant gene or predisposing gene‘functional expression’ means the expression of a gene product whosepresence gives rise to a deleterious effect or predisposes to adeleterious effect. By deleterious effect is meant giving rise to orpredisposing to disease pathology or altering the effect(s) and/orefficiency of an administered compound.

In a particular embodiment of the invention the strategy further employsribozymes that can be designed to elicit cleavage of target RNAs. Thestrategy further employs nucleotides that form triple helix DNA. Thestrategy can employ peptide nucleic acids for suppression. Nucleic acidsfor antisense, ribozymes, triple helix forming DNA and peptide nucleicacids may be modified to increase stability, binding efficiencies anduptake. Nucleic acids can be incorporated into a vector. Vectors includenaked DNA, DNA plasmid vectors, RNA or DNA virus vectors, lipids,polymers or other derivatives and compounds to aid gene delivery andexpression.

The invention further provides the use of antisense nucleotides,ribozymes, PNAs, triple helix nucleotides, or other suppressioneffectors alone or in a vector or vectors, wherein the nucleic acids areable to bind specifically or partially specifically to coding sequencesof a gene to prevent or reduce the functional expression thereof, in thepreparation of a medicament for the treatment of an autosomal dominantor polygenic disease or to increase the utility and/or action of anadministered compound.

In a further embodiment of the invention, target sequences forsuppression can include non-coding sequences of the gene.

According to the present invention there is provided a strategy forsuppressing specifically or partially specifically an endogenous geneand (if required) introducing a replacement gene, said strategycomprising the steps of:

-   1. providing nucleic acids or other suppression effectors able to    bind to an endogenous gene, gene transcript or gene product to be    suppressed and-   2. providing genomic DNA or cDNA (complete or partial) encoding a    replacement gene wherein the nucleic acids are unable to bind to    equivalent regions in the genomic DNA or cDNA to prevent expression    of the replacement gene. The replacement nucleic acids will not be    recognised by suppression nucleic acids or will be recognised less    effectively than the endogenous gene. The coding sequence of    replacement nucleic acids can be altered to prevent or reduce    efficiency of suppression. Replacement nucleic acids have    modifications in one or more third base (wobble) positions such that    replacement nucleic acids still code for the wild type or equivalent    amino acids.

In a particular embodiment of the invention there is provided a strategyfor gene suppression targeted to coding sequences of the gene to besuppressed. Suppression will be specific or partially specific to oneallele, for example, to the allele carrying a deleterious mutation.Suppressors are targeted to coding regions of a gene or to a combinationof coding and non-coding regions of a gene. Suppressors can be targetedto a characteristic of one allele of a gene such that suppression isspecific or partially specific to one allele of a gene (PCT/GB97/00574).The invention further provides for use of replacement nucleic acids withaltered coding sequences such that replacement nucleic acids will not berecognised (or will be recognised less effectively) by suppressionnucleic acids that are targeted specifically or partially specificallyto one allele of the gene to be suppressed. Replacement nucleic acidsprovide the wild type gene product, an equivalent gene product or animproved gene product but are protected completely or partially fromsuppression effectors targeted to coding sequences.

In a further embodiment of the invention, replacement nucleic acids areprovided such that replacement nucleic acids will not be recognised bynaturally occurring suppressors found to inhibit or reduce geneexpression in one or more individuals, animals or plants. The inventionprovides for use of replacement nucleic acids that have alteredsequences targeted by suppressors of the gene such that suppression bynaturally occurring suppressors is completely or partially prevented.

In an additional embodiment of the invention, there is providedreplacement nucleic acids with altered nucleotide sequence in codingregions such that replacement nucleic acids code for a product with oneor more altered amino acids. Replacement nucleic acids provide a geneproduct that is equivalent to or improved compared with the naturallyoccurring endogenous wild type gene product.

In an additional embodiment of the invention there is provided astrategy to suppress a gene where the gene transcript or gene productinterferes with the action of an administered compound.

The invention further provides the use of a vector or vectors containingsuppression effectors in the form of nucleic acids, said nucleic acidsbeing directed towards coding sequences or combinations of coding andnon-coding sequences of the target gene and vector(s) containing genomicDNA or cDNA encoding a replacement gene sequence to which nucleic acidsfor suppression are unable to bind (or bind less efficiently), in thepreparation of a combined medicament for the treatment of an autosomaldominant or polygenic disease. Nucleic acids for suppression orreplacement gene nucleic acids may be provided in the same vector or inseparate vectors. Nucleic acids for suppression or replacement genenucleic acids may be provided as a combination of nucleic acids alone orin vectors.

The invention further provides a method of treatment for a diseasecaused by an endogenous mutant gene, said method comprising sequentialor concomitant introduction of

-   (a) nucleic acids to the coding regions of a gene to be suppressed    and/or nucleic acids to coding regions and any combination of 5′    and/or 3′ untranslated regions, intronic regions, control regions or    regions adjacent to a gene to be suppressed-   (b) replacement nucleic acids with sequences that allow the    replacement gene to be expressed.

The nucleic acid for gene suppression can be administered before, afteror at the same time as the replacement gene is administered.

The invention further provides a kit for use in the treatment of adisease caused by a deleterious mutation in a gene, the kit comprisingnucleic acids for suppression able to bind to the gene to be suppressedand if required a replacement nucleic acid to replace the mutant genehaving sequence that allows it to be expressed and completely orpartially escape suppression.

Nucleotides can be administered as naked DNA or RNA. Nucleotides can bedelivered in vectors. Naked nucleic acids or nucleic acids in vectorscan be delivered with lipids or other derivatives which aid genedelivery. Nucleotides may be modified to render them more stable, forexample, resistant to cellular nucleases while still supporting RNase Hmediated degradation of RNA or with increased binding efficiencies.Antibodies or peptides can be generated to target the protein productfrom the gene to be suppressed.

The strategy described herein has applications for alleviating autosomaldominant diseases. Complete silencing of a disease allele may bedifficult to achieve using antisense, PNA, ribozyme and triple helixapproaches or any combination of gene silencing approaches. Howeversmall quantities of mutant product may be tolerated in some autosomaldominant disorders. In others a significant reduction in the proportionof mutant to normal product may result in an amelioration of diseasesymptoms. Hence, this invention may be applied to any autosomaldominantly or polygenically inherited disease in man where the molecularbasis of the disease has been established or is partially understood.This strategy enables the same therapy to be used to treat a range ofdifferent disease mutations within the same gene. The development ofsuch approaches is important to future therapies for autosomal dominantand polygenic diseases, the key to a general strategy being that itcircumvents the need for a specific therapy for every mutation causingor predisposing to a disease. This is particularly relevant in somedisorders, for example, rhodopsin linked autosomal dominant RP, in whichto date about one hundred different mutations in the rhodopsin gene havebeen observed in adRP patients. Likewise, hundreds of mutations havebeen identified in the human type I Collagen 1A1 and 1A2 genes inautosomal dominant osteogenesis imperfecta. Costs of developingtherapies for each mutation are prohibitive at present. Inventions suchas this using a general approach for therapy will be required. Generalapproaches may be targeted to the primary defect, as is the case withthis invention, or to secondary effects such as apoptosis.

This invention may be applied in gene therapy approaches forbiologically important polygenic disorders affecting large proportionsof the world's populations such as age related macular degeneration,glaucoma, manic depression, cancers having a familial component andindeed many others. Polygenic diseases require inheritance of more thanone mutation (component) to give rise to the disease state. Notably anamelioration in disease symptoms may require reduction in the presenceof only one of these components, that is, suppression of one genotypewhich, together with others leads to the disease phenotype, may besufficient to prevent or ameliorate symptoms of the disease. In somecases suppression of more than one component may be required to improvedisease symptoms. This invention may be applied in possible futureinterventive therapies for common polygenic diseases to suppress aparticular genotype(s) using suppression and, when necessary,replacement steps.

The present invention is exemplified using four genes: human rhodopsin,mouse rhodopsin, human peripherin and human collagen 1A2. The first ofthese genes are retinal specific. In contrast, collagen 1A2 is expressedin a range of tissues including skin and bone. While these four geneshave been used as examples there is no reason why the invention couldnot be deployed in the suppression of many other genes in whichmutations cause or predispose to a deleterious effect. Many examples ofmutant genes that give rise to disease phenotypes are available from theprior art—these genes all represent targets for the invention. Thepresent invention is exemplified using hammerhead ribozymes withantisense arms to elicit RNA cleavage. There is no reason why othersuppression effectors directed towards genes, gene transcripts or geneproducts could not be used to achieve gene suppression. Many examplesfrom the prior art detailing use of suppression effectors such as, interalia, antisense RNA/DNA, triple helix forming DNA, PNAs and peptides toachieve suppression of gene expression are reported. The presentinvention is exemplified using hammerhead ribozymes with antisense armsto elicit sequence specific cleavage of transcripts from genesimplicated in dominant disorders and non-cleavage of transcripts fromreplacement genes containing sequence modifications in wobble positionssuch that the replacement gene still codes for wild type protein. Thepresent invention is exemplified using suppression effectors targetingsites in coding regions of the human and mouse rhodopsin, humanperipherin and human collagen 1A2 genes. Target sites, which includesequences from transcribed but untranslated regions of genes, introns,regions involved in the control of gene expression, regions adjacent tothe gene or any combination of these, could be used to achieve genesuppression. Multiple suppression effectors, for example, shotgunribozymes could be used to optimise efficiency of suppression whennecessary. Additionally, when required, expression of a modifiedreplacement gene such that the replacement gene product is altered fromthe wild type product such that it provides a beneficial effect may beused to provide gene product.

Materials and Methods

Cloning vectors

cDNA templates and ribozymes were cloned into commercial expressionvectors (pCDNA3, pZeoSV or pbluescript) that enable expression in a testtube from T7, T3 or SP6 promoters or expression in mammalian cells fromCMV or SV40 promoters. DNA inserts were cloned into the multiple cloningsite (MCS) of these vectors typically at or near the terminal ends ofthe MCS to delete most of the MCS and thereby prevent any possibleproblems with efficiency of expression subsequent to cloning.

Sequencing Protocols

Clones containing template cDNAs and ribozymes were sequenced by ABIautomated sequencing machinery using standard protocols.

Expression of RNAs

RNA was obtained from clones by in vitro transcription using acommercially available Ribomax expression system (Promega) and standardprotocols. RNA purifications were undertaken using the Bio-101 RNApurification kit or a solution of 0.3M sodium acetate and 0.2% SDS afterisolation from polyacrylamide gels. Cleavage reactions were performedusing standard protocols with varying MgCl₂ concentrations (0-15 mM) at37° C., typically for 3 hours. Time points were performed at thepredetermined optimal MgCl₂ concentrations for up to 5 hours.Radioactively labelled RNA products were obtained by incorporating α-p³²rUTP (Amersham) in the expression reactions (Gaughan et al. 1995).Labelled RNA products were run on polyacrylamide gels before cleavagereactions were undertaken for the purpose of RNA purification andsubsequent to cleavage reactions to establish if RNA cleavage had beenachieved. Cleavage reactions were undertaken with 5 mM Tris-HCl pH8.0and varying concentrations of MgCl₂ at 37° C.

RNA Secondary Structures

Predictions of the secondary structures of human and mouse rhodopsin,human peripherin and human collagen 1A2 mRNAs were obtained using theRNAPlotFold program. Ribozymes and antisense were designed to targetareas of the RNA that were predicted to be accessible to suppressioneffectors, for example open loop structures. The integrity of open loopstructures was evaluated from the 10 most probable RNA structures.Additionally, predicted RNA structures for truncated RNA products weregenerated and the integrity of open loops between full length andtruncated RNAs compared.

Templates and Ribozymes

Human Rhodopsin

Template cDNA

The human rhodopsin cDNA (SEQ ID NO:1) was cloned into the HindIII andEcoRI sites of the MCS of pCDNA3 in a 5′ to 3′ orientation allowingsubsequent expression of RNA from the T7 or CMV promoter in the vector.The full length 5′ UTR sequence was inserted into this clone usingprimer driven PCR mutagenesis and a HindIII (in pCDNA3) to BstEII (inthe coding sequence of the human rhodopsin cDNA) DNA fragment.

cDNA with Altered Sequence at a Wobble Position

The human rhodopsin hybrid CDNA with a single base alteration (SEQ IDNO:2), a C-->G change (at nucleotide 271 of SEQ ID NO:2) was introducedinto human rhodopsin CDNA, using a HindIII to BstEII PCR cassette, byprimer directed PCR mutagenesis. This sequence change occurs at a silentposition—it does not give rise to an amino acid substitution—however iteliminates the ribozyme cleavage site (GUX -->GUG). The hybrid rhodopsinwas cloned into pCDNA3 in a 5′ to 3′ orientation allowing subsequentexpression of RNA from the T7 or CMV promoter in the vector.

Rhodopsin cDNA Carrying a Pro23Leu adRP Mutation

A human rhodopsin adRP mutation, a C-->T change (at codon 23; nucleotide217 of SEQ ID NO:3) was introduced into human rhodopsin cDNA, using aHindIII to BstEII PCR cassette by primer directed PCR mutagenesis. Thissequence change results in the substitution of a Proline for a Leucineresidue. Additionally the nucleotide change creates a ribozyme cleavagesite (CCC-->CTC) (nucleotide 216-218 of SEQ ID NO:3). The mutatedrhodopsin nucleic acid sequence was cloned into the HindIII and EcoRIsites of pCDNA3 in a 5′ to 3′ orientation allowing subsequent expressionof RNA from the T7 or CMV promoter in the vector (SEQ ID NO:3).

Ribozyme Constructs

A hammerhead ribozyme (termed Rz10 (SEQ ID NO:29) designed to target alarge conserved open loop structure in the RNA from the coding regionsof the gene was cloned subsequent to synthesis and annealing into theHindIII and XbaI sites of pCDNA3 again allowing expression of RNA fromthe T7 or CMV promoter in the vector (SEQ ID NO:4). The target site wasGUC (the GUX rule) at position 475-477 (nucleotides 369-371 of SEQ IDNO:1) of the human rhodopsin sequence. Note there is a one base mismatchin one antisense arm of Rz10. A hammerhead ribozyme (termed Rz20(SEQ IDNO:30) designed to target an open loop structure in RNA from the codingregion of a mutant rhodopsin gene with a Pro23Leu mutation was clonedsubsequent to synthesis and annealing into the HindIII and XbaI sites ofpCDNA3 again allowing expression of RNA from the T7 or CMV promoter inthe vector (SEQ ID NO:5). The target site was CTC (the NUX rule) atcodon 23 (nucleotides 216-218 of SEQ ID NO:3) of the human rhodopsinsequence (Accession number: K02281). Antisense flanks are underlined.(SEQ ID NO:29; nucleotides 101-137 of SEQ ID NO:4) Rz10:GGTCGGTCTGATGAGTCCGTGAGGACGAAACGTAGAG (SEQ ID NO:30; nucleotides 104-140of SEQ ID NO:5) Rz20: TACTCGAACTGATGAGTCCGTGAGGACGAAAGGCTGCMouse RhodopsinTemplate cDNA

The full length mouse rhodopsin CDNA was cloned into the EcoRI sites ofthe MCS of pCDNA3 in a 5′ to 3′ orientation allowing subsequentexpression of RNA from the T7 or CMV promoter in the vector (SEQ IDNO:6).

cDNA with Altered Sequence at a Wobble Position

The mouse rhodopsin hybrid cDNA with a single base alteration, a T-->Cchange (at position 1460) (nucleotide 190 of SEQ ID NO:7) was introducedinto mouse rhodopsin cDNA, using a HindIII to Eco47III PCR cassette, byprimer directed PCR mutagenesis. This sequence change occurs at a silentposition—it does not give rise to an amino acid substitution—however iteliminates the ribozyme cleavage site (TTT-->TCT) (nucleotides 189-191of SEQ ID NO:7). The hybrid rhodopsin was cloned into pCDNA3 in a 5′ to3′ orientation allowing subsequent expression of RNA from the T7 or CMVpromoter in the vector (SEQ ID NO:7).

Ribozyme Constructs

A hammerhead ribozyme (termed Rz33) (SEQ ID NO:31) designed to target alarge robust open loop structure in the RNA from the coding regions ofthe gene was cloned subsequent to synthesis and annealing into theHindIII and XbaI sites of pCDNA3 again allowing expression of RNA fromthe T7 or CMV promoter in the vector (SEQ ID NO:8). The target site wasTTT (the NUX rule) at position 1459-1461 (nucleotides 405-407 of SEQ IDNO:6) of the mouse rhodopsin sequence. (Accession number: M55171).Antisense flanks are underlined. (SEQ ID NO:31; nucleotides 118-154 ofSEQ ID NO:8) Rz33: GGCACATCTGATGAGTCCGTGAGGACGAAAAAATTGGHuman PeripherinTemplate cDNA

The full length human peripherin cDNA was cloned into the EcoRI sites ofthe MCS of pCDNA3 in a 5′ to 3′ orientation allowing subsequentexpression of RNA from the T7 or CMV promoter in the vector (SEQ IDNO:9).

DNAs with Altered Sequence at a Wobble Position

A human peripherin hybrid DNA with a single base alteration, a A-->Gchange (at position 257) (nucleotide 332 of SEQ ID NO:10) was introducedinto human peripherin DNA by primer directed PCR mutagenesis (forward257 mutation primer—5′CATGGCGCTGCTGAAAGTCA3′ (SEQ ID NO:11)—the reverse257 primer was complementary to the forward primer). This sequencechange occurs at a silent position—it does not give rise to an aminoacid substitution—however it eliminates the ribozyme cleavage site(CTA-->CTG)(nucleotide 330-332 of SEQ ID NO:10). A second humanperipherin hybrid DNA with a single base alteration, a A-->G change (atposition 359) (nucleotide 468 of SEQ ID NO:13) was introduced into humanperipherin DNA, again by primer directed PCR mutagenesis (forward 359mutation primer—5′CATCTTCAGCCTGGGACTGT3′ (SEQ ID NO:12)—the reverse 359primer was complementary to the forward primer) (SEQ ID NO:12).Similarly this sequence change occurs at a silent position—it does notgive rise to an amino acid substitution—however again it eliminates theribozyme cleavage site (CTA-->CTG) (nucleotides 466-468 of SEQ IDNO:13). The ribozyme cleavage sites at 255-257 (nucleotides 330-332 ofSEQ ID NO:10) and 357-359 (nucleotides 466-468 of SEQ ID NO:13) occur atdifferent open loop structures as predicted by the RNAPlotFold program.Hybrid peripherin DNAs included the T7 promoter sequence allowingsubsequent expression of RNA.

Ribozyme Constructs

Hammerhead ribozymes (termed Rz30 and Rz31)(SEQ ID NOs: 32 and 33,respectively), designed to target robust open loop structures in the RNAfrom the coding regions of the gene, were cloned subsequent to synthesisand annealing into the HindIII and XbaI sites of pCDNA3 again allowingexpression of RNA from the T7 or CMV promoter in the vector (SEQ IDNOS:14 and 15, respectively). The target sites were both CTA (the NUXrule) at positions 255-257 and 357-359 respectively of the humanperipherin sequence. (Accession number: M73531). Antisense flanks areunderlined. (SEQ ID NO:32; nucleotides 116-153 of SEQ ID NO:14) Rz30:ACTTTCAGCTGATGAGTCCGTGAGGACGAAAGCGCCA (SEQ ID NO:33; nucleotides 112-148of SEQ ID NO:15) Rz31: ACAGTCCCTGATGAGTCCGTGAGGACGAAAGGCTGAAHuman Type I Collagen—COL1A2Template cDNA

A human type I collagen 1A2 cDNA was obtained from the ATCC (AccessionNo: Y00724). A naturally occurring polymorphism has previously beenfound in collagen 1A2 at positions 907 of the gene involving a T-->Anucleotide change (Filie et al. 1993). The polymorphism occurs in apredicted open loop structure of human collagen 1A2 RNA. Polymorphicvariants of human collagen 1A2 were generated by PCR directedmutagenesis, using a HindIII to XbaI PCR cassette. Resulting clonescontained the following polymorphism : collagen 1A2 (A) has a Tnucleotide at position 907 (A nucleotide 176 of SEQ ID NO:17, reversestrand). In contrast human collagen 1A2 (B) has an A nucleotide atposition 907 (T nucleotide 181 of SEQ ID NO:16, reverse strand). Incollagen 1A2 (A) there is a ribozyme target site, that is a GTC site(906-908) (nucleotides 175-177 of SEQ ID NO:17, reverse strand), howeverthis cleavage site is lost in collagen 1A2 (B) as the sequence isaltered to GAC (906-908) (nucleotides 180-182 of SEQ ID NO:16, reversestrand), thereby disrupting the ribozyme target site.

Ribozyme Constructs

A hammerhead ribozyme (termed Rz907) (SEQ ID NO:34) was designed totarget a predicted open loop structure in the RNA from the coding regionof the polymorphic variant of the human collagen 1A2 gene. Rz907oligonucleotide primers were synthesised, annealed and cloned into theHindIII and XbaI sites of pCDNA3 again allowing subsequent expression ofRNA from the T7 or CMV promoter in the vector (SEQ ID NO:18). The targetsite was a GUX site at position 906-908 of the human type I collagen 1A2sequence (Accession number: Y00724). Antisense flanks are underlined.(SEQ ID NO:34; nucleotide 107-141 of SEQ ID NO:18) Rz907:CGGCGGCTGATGAGTCCGTGAGGACGAAACCAGCA

FIGURE LEGENDS

FIG. 1:

pBR322 was cut with MspI, radioactively labelled and run on apolyacrylamide gel to enable separation of the resulting DNA fragments.The sizes of these fragments are given in FIG. 1. This DNA ladder wasthen used on subsequent polyacrylamide gels (4-8%) to provide anestimate of the size of the RNA products run on the gels. However thereis a significant difference in mobility between DNA and RNA depending onthe percentage of polyacrylamide and the gel running conditions—hencethe marker provides an estimate of size of transcripts.

FIG. 2:

-   A: Human rhodopsin CDNA (SEQ ID NO:1) was expressed from the T7    promoter to the BstEII site in the coding sequence. Resulting RNA    was mixed with RZ10RNA in 15 mM magnesium chloride and incubated at    37° C. for varying times. Lanes 1-4: Human rhodopsin RNA and Rz10RNA    after incubation at 37° C. with 15 mM magnesium chloride for 0, 1 2    and 3 hours respectively. Sizes of the expressed RNAs and cleavage    products are as expected (Table 1). Complete cleavage of human    rhodopsin RNA was obtained with a small residual amount of intact    RNA present at 1 hour. Lane 6 is intact unadapted human rhodopsin    RNA (BstEII) alone. Lane 5 is unadapted human rhodopsin RNA (FspI)    alone and refers to FIG. 2B. From top to bottom, human rhodopsin RNA    and the two cleavage products from this RNA are highlighted with    arrows.-   B: The unadapted human rhodopsin cDNA was expressed from the T7    promoter to the FspI site in the coding sequence. The adapted human    rhodopsin cDNA was expressed from the T7 promoter to the BstEII site    in the coding sequence. Lanes 1-4: Resulting RNAs were mixed    together with Rz10 and 15 mM magnesium chloride and incubated at    37° C. for varying times (0, 1 , 2 and 3 hours respectively). The    smaller unadapted rhodopsin transcripts were cleaved by Rz10 while    the larger adapted transcripts were protected from cleavage by Rz10.    Cleavage of adapted protected transcripts would have resulted in    products of 564bases and 287bases—the 564bases product clearly is    not present—the 287bp product is also generated by cleavage of the    unadapted human rhodopsin transcripts and hence is resent (FspI).    After 3 hours the majority of the unadapted rhodopsin transcripts    has been cleaved by Rz10. Lane 5 contains the intact adapted human    rhodopsin RNA (BstEII) alone. From top to bottom adapted uncleaved    human rhodopsin transcripts, residual unadapted uncleaved human    rhodopsin transcripts and the larger of the cleavage products from    unadapted human rhodopsin transcripts are highlighted by arrows. The    smaller 22 bases cleavage product from the unadapted human rhodopsin    transcripts has run off the gel.

FIG. 3:

-   A: Unadapted (SEQ ID NO:l) and adapted (SEQ ID NO:2) human rhodopsin    cDNAs were expressed from the T7 promoter to the AcyI after the    coding sequence and the BstEII site in the coding sequence    respectively. Sizes of expressed RNAs and cleavage products were as    predicted (Table 1). Resulting RNAs were mixed together with Rz10    RNA at varying magnesium chloride concentrations and incubated at    37° C. for 3 hours. Lane 1: Intact unadapted human rhodopsin RNA    (AcyI) alone. Lanes 2-5: Unadapted and adapted human rhodopsin RNAs    and Rz10 RNA after incubation at 37° C. with 0, 5, 10 and 15 mM    MgCl₂ respectively. Almost complete cleavage of the larger unadapted    human rhodopsin RNA was obtained with a small residual amount of    intact RNA present at 5 mM MgCl₂. In contrast the adapted human    rhodopsin RNA remained intact. From top to bottom, the unadapted and    adapted rhodopsin RNAs, and two cleavage products from the unadapted    human rhodopsin RNA are highlighted by arrows. Lane 6 is intact    adapted human rhodopsin RNA (BstEII) alone.-   B: The adapted human rhodopsin CDNA was expressed from the T7    promoter to the BstEII site in the coding sequence. Lanes 1-4:    Resulting RNA was mixed together with Rz10 and 0, 5, 10 and 15 mM    magnesium chloride and incubated at 37° C. for 3 hours respectively.    The adapted rhodopsin transcripts were not cleaved by Rz10. Cleavage    of adapted transcripts would have resulted in cleavage products of    564 bases and 287 bases which clearly are not present. Lane 5:    intact adapted human rhodopsin RNA (BstEII) alone. Lane 4: RNA is    absent—due to a loading error or degradation. The adapted uncleaved    human rhodopsin RNA is highlighted by an arrow.-   C: Unadapted (SEQ ID NO:1) and adapted (SEQ ID NO:2) human rhodopsin    cDNAs were expressed from the T7 promoter to the AcyI after the    coding sequence and the BstEII site in the coding sequence    respectively. Sizes of expressed RNAs and cleavage products were as    predicted (Table 1). Resulting RNAs were mixed together with Rz10    RNA at varying magnesium chloride concentrations and incubated at    37° C. for 3 hours. Lane 1: DNA ladder as in FIG. 1. Lanes 2-5:    Unadapted and adapted human rhodopsin RNAs and Rz10 RNA after    incubation at 37° C. with 0, 5, 10 and 15 mM MgCl₂ respectively.    Almost complete cleavage of the larger unadapted human rhodopsin RNA    was obtained with a small residual amount of intact RNA present at 5    and 10 mM MgCl₂. In contrast the adapted human rhodopsin RNA    remained intact. Lane 6: Adapted human rhodopsin RNA (BstEII) alone.    Lane 7: Unadapted human rhodopsin RNA (AcyI) alone. Lane 8: DNA    ladder as in FIG. 1. From top to bottom, the unadapted and adapted    rhodopsin RNAs, and two cleavage products from the unadapted human    rhodopsin RNA are highlighted by arrows. Separation of the adapted    human rhodopsin RNA (851 bases) and the larger of the cleavage    products from the unadapted RNA (896 bases) is incomplete in this    gel (further running of the gel would be required to achieve    separation)—however the separation of these two RNAs is demonstrated    in FIG. 3A.

FIG. 4:

The mutant (Pro23Leu) (SEQ ID NO:3) human rhodopsin cDNA was expressedfrom the T7 promoter to the BstEII in the coding sequence. Likewise theRz20 clone was expressed to the XbaI site. Resulting RNAs were mixedtogether with 10 mM magnesium chloride concentrations at 37° C. forvarying times. Sizes of expressed RNAs and cleavage products were aspredicted (Table 1). Lane 1: DNA ladder as in FIG. 1. Lanes 2: Pro23Leuhuman rhodopsin RNA alone. Lanes 3-7 Pro23Leu human rhodopsin RNA andRz20 RNA after incubation at 37° C. with 10 mM MgCl₂ for 0 mins, 30mins, 1 hr, 2 hrs and 5 hrs respectively. Almost complete cleavage ofmutant rhodopsin transcripts was obtained with a residual amount ofintact RNA left even after 5 hours. Lane 8: DNA ladder as in FIG. 1.From top to bottom, intact mutant rhodopsin RNA and the two cleavageproducts from the mutant human rhodopsin RNA are highlighted by arrows.

FIG. 5:

The mutant (Pro23Leu) (SEQ ID NO:3) human rhodopsin cDNA was expressedfrom the T7 promoter to the BstEII in the coding sequence. Likewise theRz10 clone (SEQ ID NO:4) was expressed to the XbaI site. Resulting RNAswere mixed together with 10 mM magnesium chloride concentrations at 37°C. for varying times. Sizes of expressed RNAs and cleavage products wereas predicted (Table 1). Lane 1: DNA ladder as in FIG. 1. Lanes 2:Pro23Leu human rhodopsin RNA alone. Lanes 3-7 Pro23Leu human rhodopsinRNA and Rz10 RNA after incubation at 37° C. with 10 mM MgCl₂ for 0 mins,30 mins, 1 hr, 2 hrs and 5 hrs respectively. Almost complete cleavage ofmutant human rhodopsin RNA was obtained with a residual amount of intactRNA remaining even after 5 hours (Lane 7). Lane 8: DNA ladder as inFIG. 1. From top to bottom, intact mutant rhodopsin RNA and the twocleavage products from the mutant human rhodopsin RNA are highlighted byarrows.

FIG. 6:

The mouse rhodopsin cDNA clone was expressed in vitro from the T7promoter to the Eco47III site in the coding sequence. Likewise the Rz33clone was expressed to the XbaI site. A: Resulting RNAs were mixedtogether with 10 mM magnesium chloride at 37° C. for varying times.Sizes of expressed RNAs and cleavage products were as predicted (Table1). DNA ladder as in FIG. 1. Lane 1: mouse rhodopsin RNA alone. Lanes2-5 Mouse rhodopsin RNA and Rz33 RNA after incubation at 37° C. with 10mM MgCl₂ at 0, 5, 7.5 and 10 mM MgCl₂ respectively for 3 hours. Cleavageof mouse rhodopsin RNA was obtained after addition of divalent ions(Lane 3). Residual uncleaved mouse rhodopsin RNA and the two cleavageproducts from the mouse rhodopsin RNA are highlighted by arrows. B: Theadapted mouse rhodopsin cDNA clone with a base change at position 1460(nucleotide 190 of SEQ ID NO:7) was expressed in vitro from the T7promoter to the Eco47III site in the coding sequence. Likewise the Rz33clone was expressed to the XbaI site. Resulting RNAs were mixed togetherwith various magnesium chloride concentrations at 37° C. for 3 hours.Sizes of expressed RNAs and cleavage products were as predicted (Table1). Lane 1: DNA ladder as in FIG. 1. Lane 2: Adapted mouse rhodopsin RNAalone. Lanes 3-6: Adapted mouse rhodopsin RNA and Rz33 RNA afterincubation at 37° C. with 0, 5, 7.5 and 10 mM MgCl₂ for 3 hours at 37°C. No cleavage of the adapted mouse rhodopsin RNA was observed. Theintact adapted mouse rhodopsin RNA is highlighted by an arrow.

FIG. 7:

The human peripherin cDNA clone was expressed in vitro from the T7promoter to the BglII site in the coding sequence. Likewise Rz30(targeting a cleavage site at 255-257) was expressed to the XbaI site.A: Resulting RNAs were mixed together with 10 mM magnesium chloride at37° C. for varying times. Lane 1: DNA ladder as in FIG. 1. Lane 2:Intact human peripherin RNA alone. Lanes 3-7: Human peripherin RNA andRz30 RNA after incubation at 37° C. with 10 mM MgCl₂ for 3 hrs, 2 hrs,1hr, 30 mins and 0 mins respectively. Lane 8: DNA ladder as in FIG. 1.From top to bottom, intact human peripherin RNA and the two cleavageproducts from the human peripherin RNA are highlighted by arrows. B:Resulting RNAs were mixed with Rz30 RNA at varying magnesium chlorideconcentrations and incubated at 37° C. for 3 hrs. Lane 1: DNA ladder asin FIG. 1. Lanes 2-5: Human peripherin RNA and Rz30 after incubation at37° C. with 10, 7.5, 5 and 0 mM magnesium chloride respectively for 3hrs. Lane 6: Intact human peripherin RNA alone. Sizes of the expressedRNAs and cleavage products are as expected (Table 1). Significantcleavage of human peripherin RNA was obtained with a residual amount ofintact RNA present at each MgCl₂ concentration. From top to bottom,human peripherin RNA and the two cleavage products from this RNA arehighlighted with arrows. C: The adapted human peripherin DNA with asingle base change at position 257 was expressed from the T7 promoter tothe AvrII site in the coding sequence. Resulting RNA was mixed with Rz30at various magnesium chloride concentrations and incubated at 37° C. for3 hrs. Lane 1: DNA ladder as in FIG. 1. Lane 2: Intact adapted humanperipherin RNA alone. Lanes 3-6: Adapted human peripherin RNA and Rz30after incubation at 37° C. with 0, 5, 7.5 and 10 mM magnesium chloriderespectively for 3 hrs. Lane 7: DNA ladder as in FIG. 1. D: Theunadapted human peripherin cDNA and the adapted human peripherin DNAfragment with a single base change at position 257 were expressed fromthe T7 promoter to the BglII and AvrII sites respectively in the codingsequence. Resulting RNAs were mixed with Rz30 at various magnesiumchloride concentrations and incubated at 37° C. for 3 hrs. Lane 1: DNAladder as in FIG. 1. Lane 2: Intact unadapted human peripherin RNAalone. Lane 3: Intact adapted human peripherin RNA alone. Lanes 4-7:Unadapted and adapted human peripherin RNAs and Rz30 after incubation at37° C. with 0, 5, 7.5 and 10 mM magnesium chloride respectively for 3hrs at 37° C. No cleavage of the adapted human peripherin RNA wasobserved even after 3 hours whereas a significant reduction in theunadapted RNA was observed over the same time frame. Lane 8: DNA ladderas in FIG. 1. From top to bottom, residual unadapted human peripherinRNA, adapted human peripherin RNA and the two cleavage products arehighlighted by arrows.

FIG. 8:

Human peripherin cDNA clone was expressed in vitro from the T7 promoterto the BglII site in the coding sequence. Likewise the Rz31 (targeting acleavage site at 357-359) (nucleotides 466-468 of SEQ ID NO:13) wasexpressed to the XbaI site. A: Resulting RNAs were mixed together with10 mM magnesium chloride at 37° C. for varying times. Lane 1: DNA ladderas in FIG. 1. Lanes 2-6: Human peripherin RNA and Rz31 RNA afterincubation at 37° C. with 10 mM MgCl₂ for 3 hrs, 2 hrs, 1 hr, 30 minsand 0 mins respectively. Increased cleavage of mouse rhodopsin RNA wasobtained over time—however significant residual intact RNA remained evenafter 3 hours (Lane 2). Lane 7: Intact human peripherin RNA alone. Lane8: DNA ladder as in FIG. 1. From top to bottom, intact human peripherinRNA and the two cleavage products from the human peripherin RNA arehighlighted by arrows. B: Resulting RNAs were mixed with Rz31 RNA atvarying magnesium chloride concentrations and incubated at 37° C. for 3hrs. Lane 1: DNA ladder as in FIG. 1. Lanes 2-5: Human peripherin RNAand Rz31 after incubation at 37° C. with 10, 7.5, 5 and 0 mM magnesiumchloride respectively for 3 hrs. Sizes of the expressed RNAs andcleavage products are as expected (Table 1). Significant cleavage ofhuman peripherin RNA was obtained with a residual amount of intact RNApresent at each MgCl₂ concentration (Lanes 2-4). Lane 6: Intact humanperipherin RNA alone. Lane 7: DNA ladder as in FIG. 1. From top tobottom, human peripherin RNA and the two cleavage products from this RNAare highlighted with arrows. C: The adapted human peripherin DNA with asingle base change at position 359 (nucleotide 468 of SEQ ID NO:13) wasexpressed from the T7 promoter to the BglII site in the coding sequence.Resulting RNA was mixed with Rz31 at various magnesium chlorideconcentrations and incubated at 37° C. for 3 hrs. Lane 1: DNA ladder asin FIG. 1. Lane 2: Intact adapted human peripherin RNA alone. Lanes 3-6:Adapted human peripherin RNA and RZ31 after incubation at 37° C. with 0,5, 7.5 and 10 mM magnesium chloride respectively for 3 hrs. No cleavageof the adapted human peripherin RNA was observed even after 3 hours.Lane 7: DNA ladder as in FIG. 1.

FIG. 9:

-   A: The human collagen 1A2 cDNA clones containing the A and T alleles    of the polymorphism at position 907 were expressed from the T7    promoter to the MvnI and XbaI sites in the insert and vector    respectively. Resulting RNAs were mixed together with Rz907 and    various MgCl₂ concentrations and incubated at 37° C. for 3 hours.    Lane 1: intact RNA from the human collagen 1A2 (A) containing the A    allele of the 907 polymorphism. Lane 2: intact RNA from the human    collagen 1A2 (B) containing the T allele of the 907 polymorphism.    Lanes 3-5: Human collagen 1A2 (A) and (B) representing the A and T    allele RNAs and Rz907 incubated with 0, 5, and 10 mM MgCl₂ at 37° C.    for 3 hours. RNA transcripts from the T allele containing the    906-908 target site are cleaved by Rz907 upon addition of divalent    ions—almost complete cleavage is obtained with a residual amount of    transcript from the T allele remaining (Lane 5). In contrast    transcripts expressed from the A allele (which are smaller in size    to distinguish between the A (MvnI) and T (XbaI) alleles) were not    cleaved by Rz907—no cleavage products were observed. From top to    bottom, RNA from the T allele, the allele and the two cleavage    products from the T allele are highlighted by arrows. Lane 6: DNA    ladder as in FIG. 1.-   B: The human collagen 1A2 CDNA (A)+(B) clones containing the A and T    alleles of the polymorphism at 907 were expressed from the T7    promoter to the MvnI and XbaI sites in the insert and vector    respectively. Resulting RNAs were mixed together with Rz907 and 10    mM magnesium chloride and incubated at 37° C. for varying times.    Lane 1: DNA ladder as in FIG. 1. Lane 2: intact RNA from the human    collagen 1A2 (A) with the A allele of the 907 polymorphism. Lane 3:    intact RNA from the human collagen 1A2 (B) with the T allele of the    907 polymorphism. Lanes 4-9: Human collagen 1A2 A and T allele RNA    and Rz907 incubated with10 mM MgCl₂ at 37° C. for 0, 30 mins, 1    hour, 2 hours, 3 hours and 5 hours respectively. RNA transcripts    from the T allele containing the 906-908 target site are cleaved by    Rz907—almost complete cleavage is obtained after 5 hours. In    contrast transcripts expressed from the A allele (which are smaller    in size to distinguish between the A (MvnI) and T (XbaI) alleles)    were not cleaved by Rz907—no cleavage products were observed. From    top to bottom, RNA from the T allele, the A allele and the two    cleavage products from the T allele are highlighted by arrows. FIG.    10:

The human rhodopsin cDNA in pcDNA3. (SEQ ID NO: 1).

FIG. 11:

The human rhodopsin cDNA in pcDNA3 (SEQ ID NO:2) with a base change at asilent site (477) (nucleotide 271 of SEQ ID NO:2).

FIG. 12:

Mutant (Pro23Leu) (nucleotides 216-218 of SEQ ID NO:3) human rhodopsincDNA in pcDNA3 (SEQ ID NO:3).

FIG. 13:

Rz10 cloned into pcDNA3 (SEQ ID NO:4). Note there is a one base mismatchin one antisense arm of Rz10.

FIG. 14:

Rz20cloned into pcDNA3 (SEQ ID NO:5).

FIG. 15:

The mouse rhodopsin cDNA in pcDNA3 (SEQ ID NO:6).

FIG. 16:

The mouse rhodopsin cDNA in pcDNA3 (SEQ ID NO:7) with a base change at asilent site (1460) (nucleotide 190 of SEQ ID NO:7).

FIG. 17:

Rz33 cloned into pcDNA3 (SEQ ID NO:8)

FIG. 18:

The human peripherin CDNA in pcDNA3 (SEQ ID NO:9).

FIG. 19:

The human peripherin DNA fragment (SEQ ID NO:10) with a base change at asilent site (257) (nucleotide 332 of SEQ ID NO:10).

FIG. 20:

The human peripherin DNA fragment (SEQ ID NO:11) with a base change at asilent site (359) (nucleotide 468 of SEQ ID NO:13). The sequence qualitywas not good in the region of the human peripherin 359 silent change(nucleotide 468 of SEQ ID NO:13)—the sequencing primer was too far fromthe target site to achieve good quality sequence.

FIG. 21:

Rz30 cloned into pcDNA3 (SEQ ID NO:12)

FIG. 22:

Rz31 cloned into pcDNA3 (SEQ ID NO:13)

FIG. 23:

Collagen 1A2 (A) sequence containing the A polymorphism at position 907.(SEQ ID NO:14) (Note there is an additional polymorphic site at position902).

FIG. 24:

Collagen 1A2 (B) sequence containing the T polymorphism at position 907.(SEQ ID NO:15) (Note there is an additional polymorphic site at position902).

FIG. 25:

Rz907 cloned into pcDNA3 (SEQ ID NO:18)

RESULTS

Human and mouse rhodopsin, human peripherin and human collagen 1A2 cDNAclones were expressed in vitro. Ribozymes targeting specific sites inthe human and mouse rhodopsin, human peripherin and human collagen 1A2cDNAs were also expressed in vitro. cDNA clones were cut with variousrestriction enzymes resulting in the production of differently sizedtranscripts after expression. This aided in differentiating between RNAsexpressed from unadapted and adapted cDNAs. Restriction enzymes used tocut each clone, sizes of resulting transcripts and predicted sizes ofproducts after cleavage by target ribozymes are given below in Table 1.Exact sizes of expression products may vary by a few bases from thatestimated as there may be some ambiguity concerning inter alia thespecific base at which transcription starts.

EXAMPLE 1

A: Human Rhodopsin

The unadapted human rhodopsin CDNA (SEQ ID NO:1) and the human rhodopsinCDNA with a single nucleotide substitution in the coding sequence (SEQID NO:2) were cut with BstEII and expressed in vitro. The single basechange occurs at the third base position or wobble position of the codon(at position 477) (nucleotide 271 of SEQ ID NO:2) and therefore does notalter the amino acid coded by this triplet. The Rz10 clone was cut withXbaI and expressed in vitro. Resulting ribozyme and human rhodopsin RNAswere mixed with varying concentrations of MgCl₂ to optimise cleavage oftemplate RNA by Rz10. A profile of human rhodopsin RNA cleavage by Rz10over time is given in FIG. 2A. The MgCl₂ curve profile used to test ifadapted human rhodopsin transcripts could be cleaved by Rz10 is given inFIG. 3B. Unadapted and adapted human rhodopsin cDNAs were cut with FspIand BstEII respectively, expressed and mixed together with Rz10 RNA totest for cleavage (FIG. 2B) over time. Likewise, unadapted and adaptedhuman rhodopsin cDNAs were cut with AcyI and BstEII respectively, bothwere expressed in vitro and resulting transcripts mixed with Rz10 RNA atvarying MgCl₂ concentrations to test for cleavage (FIG. 3A, 3C). In allcases expressed RNAs were the predicted size.

Similarly in all cases unadapted transcripts were cleaved into productsof the predicted size. Cleavage of nadapted human rhodopsin RNA wasalmost complete—little residual uncleaved RNA remained. In all casesadapted human rhodopsin RNAs with a single base change at a silent siteremained intact, that is, they were not cleaved by Rz10. Clearly,transcripts from the unadapted human rhodopsin cDNA are cleaved by Rz10while transcripts from the adapted replacement gene which has beenmodified in a manner which exploits the degeneracy of the genetic codeare protected from cleavage. It is worth noting that AcyI enzyme cutsafter the stop codon and therefore the resulting RNA includes thecomplete coding sequence of the gene.

B: Human Rhodopsin

Rz20 was cut with XbaI and expressed in vitro. Similarly the rhodopsincDNA containing a Pro23Leu mutation was cut with BstEII and expressed invitro. Resulting RNAs were mixed and incubated at 37° C. with 10 mMMgCl₂ for varying times. Rz20 was designed to elicit mutation specificcleavage of transcripts containing a Pro23Leu rhodopsin mutation. Allexpressed products and cleavage products were the correct size. FIG. 4demonstrates mutation specific cleavage of the mutant RNA over timeincubated at 37° C. with 10 mM MgCl₂. Cleavage of mutant rhodopsintranscripts by Rz10 which targets a ribozyme cleavage site 3′ of thesite of the Pro23Leu mutation in rhodopsin coding sequence was explored.The mutant rhodopsin cDNA and Rz10 clones were cut with BstEII and XbaIrespectively and expressed in vitro. Resulting RNAs were mixed andincubated with 10 mM MgCl₂ for varying times (FIG. 5). All expressedproducts and cleavage products were the correct size. Rz10 cleavedmutant rhodopsin transcripts. Using a replacement gene with a sequencechange around the Rz10 cleavage site which is at a wobble position wedemonstrated in Example 1A that transcripts from the replacement generemain intact due to absence of the Rz10 target site (FIGS. 2B, 3A and3B). Hence Rz10 could be used to cleave mutant transcripts in a mannerindependent of the disease mutation itself (that is, using this site)while transcripts from the replacement gene which code for the correctprotein would remain intact and therefore could supply the wild typeprotein.

Example 2 Mouse Rhodopsin

Rz33 was cut with XbaI and expressed in vitro. Similarly the mouserhodopsin cDNA was cut with Eco47III and expressed in vitro. ResultingRNAs were mixed and incubated with varying concentrations of MgCl₂. Allexpressed products and cleavage products were the correct size. FIG. 6Ademonstrates specific cleavage of the mouse rhodopsin RNA over variousMgCl₂ concentrations incubated at 37° C. for 3 hours. Using areplacement gene with a sequence change around the Rz33 cleavage site(TTT-->TCT) (nucleotides 189-191 of SEQ ID NO:7) which is at a wobbleposition we demonstrated that transcripts from the replacement generemain intact due to absence of the Rz33 target site (FIG. 6B). HenceRz33 could be used to cleave mutant transcripts in a manner independentof the disease mutation itself (that is, using this site) whiletranscripts from the replacement gene which code for the correct proteinwould remain intact and therefore could supply the wild type protein.

Example 3 Human Peripherin

The unadapted human peripherin cDNA and two human peripherin DNAfragments generated by PCR mutagenesis with a single nucleotidesubstitution in the coding sequence were cut with BglII and AvrIIrespectively and expressed in vitro. The single base changes in theadapted DNAs occur at third base positions or wobble positions of thecodon (at position 257 and 359) (nucleotide 468 of SEQ ID NO:13 andnucleotide 332 of SEQ ID NO:10, respectively) and therefore do not alterthe amino acid coded by these triplets. The Rz30 and Rz31 clones werecut with XbaI and expressed in vitro. Resulting ribozymes and unadaptedhuman rhodopsin RNAs were mixed with varying concentrations of MgCl₂ tooptimise cleavage of template RNA by Rz30 and Rz31 . Profiles of humanperipherin RNA cleavage by Rz30 over various MgCl₂ concentrations andover time are given in FIG. 7. Similarly profiles of human peripherinRNA cleavage by Rz31 over various MgCl₂ concentrations and over time aregiven in FIG. 8. In all cases expressed RNAs were the predicted size.Similarly in all cases unadapted transcripts were cleaved into productsof the predicted size. Adapted human rhodopsin RNAs were mixed togetherwith Rz30 and Rz31 RNA over various MgCl₂ concentrations to test ifadapted human peripherin transcripts could be cleaved by Rz30 and Rz31(FIGS. 7 and 8). Expressed RNAs were the predicted size. In all casesadapted human peripherin RNAs with single base changes at silent sitesremained intact, that is, they were not cleaved by Rz30 or Rz31.Clearly, transcripts from the unadapted human peripherin cDNA arecleaved by Rz30 and Rz31 while transcripts from the adapted replacementDNAs which have been modified in a manner which exploits the degeneracyof the genetic code are protected from cleavage.

Example 4 Human Collagen 1A2

Rz907 clones targeting a polymorphic site in human collagen 1A2 sequencewas cut with XbaI and expressed in vitro. The human collagen 1A2 cDNAclones (A and B) containing two allelic forms of a polymorphism in thecoding sequence of the gene at positions 907 were cut with MvnI and XbaIrespectively, expressed in vitro and RNAs mixed together with Rz907 RNAto test for cleavage of transcripts by this ribozyme. All expressedtranscripts were of the predicted sizes. RNAs were mixed with varyingconcentrations of MgCl₂ to optimise cleavage of RNAs by Rz907 (FIG. 9).Notably the majority of the RNA transcripts from human collagen 1A2 (A)which has a T nucleotide at position 907 (A nucleotide 176 of SEQ IDNO:17, reverse strand) is cleaved by Rz907 (FIG. 9).

This allelic form of the gene has a ribozyme cleavage site at 906-908.Notably the situation is reversed with transcripts from human collagen1A2 (B) where in this allelic form of the gene due to the nature of thepolymorphism present at position 907 the ribozyme cleavage site has beenlost.

In contrast to transcripts from human collagen (A), transcripts fromhuman collagen (B) were protected from cleavage by Rz907 due to thealteration in the sequence around the ribozyme cleavage site (FIG. 9).Cleavage of collagen 1A2 (A) by Rz907 was efficient which is consistentwith 2-D predictions of RNA open loop structures for the polymorphism—inthe allele containing the Rz907 ribozyme cleavage site, the target siteis found quite consistently in an open loop structure. This polymorphismfound in an open loop structure of the transcript clearly demonstratesthe feasibility and utility of using the degeneracy of the genetic codein the suppression of an endogenous gene (either suppressing bothalleles or a single allele at a polymorphic site) and restoration ofgene expression using a gene which codes for the same protein but hassequence modifications at third base wobble positions which protect thereplacement gene from suppression. TABLE 1 Restriction Cleavage EnzymeRNA Size Products Example 1 Human rhodopsin BstEII 851 bases 287 + 564bases AcyI 1183 bases 287 + 896 bases FspI 309 bases 287 + 22 AdaptedHuman BstEII 851 bases rhodopsin Human rhodopsin BstEII 851 bases 170 +681 (Rz20) Pro-Leu Human rhodopsin BstEII 851 bases 287 + 564 (Rz10)Pro-Leu Rz10 XbaI 52 bases Rz20 XbaI 52 bases (Table 1; SEQ ID NOS: 1-5;FIGS. 1-5) Example 2 Mouse rhodopsin Eco47III 774 bases 400 + 374Adapted mouse Eco47III 774 bases rhodopsin Rz33 XbaI 52 bases (Table 1;SEQ ID NOS: 6-9; FIG. 6) Example 3 Human peripherin BglII 545 bases315 + 230 (Rz30) Human peripherin BglII 545 bases 417 + 128 (Rz31)Adapted human AvrII 414 bases peripherin Adapted human BglII 545 basesperipherin Rz30 XbaI 52 bases Rz31 XbaI 52 bases (Table 1; SEQ ID NOS:10, 13-16; FIGS. 7 and 8) Example 4 Human Collagen 1A2 XbaI 888 bases690 + 198 bases (B) -Rz907 Human Collagen MvnI 837 bases 1A2 (A) Rz907XbaI 52 bases (Table 1; SEQ ID NOS: 16-18; FIG. 9)

TABLE 2 A: Rhodopsin mutations tested to assess if the predicted openloop RNA structure containing the Rz10 target site (475-477) remainsintact in mutant transcripts. Rhodopsin mutation RNA open loop targetedby Rz10 Pro 23 Leu Intact Gly 51 Val Intact Thr 94 Ile Intact Gly 188Arg Intact Met 207 Arg Intact Ile del 255 Intact B: Utilisation of thedegeneracy of the genetic code. Ribozyme cleavage sites are underlineduz,1/11 Human rhodopsin Unadapted sequence       475-477 TAC GTC ACC GTCCAG (SEQ ID NO:19)       Val Adapted sequence       475-477 TAC GTG ACCGTC CAG (SEQ ID NO:20)       Val Mouse rhodopsin Unadapted sequence 1459-1461 AAT TTT TAT GTG CCC (SEQ ID NO:21)      Phe Adapted sequence 1459-1461 AAT TTC TAT GTG CCC (SEQ ID NO:22)      Phe Human peripherinUnadapted sequence    255-257 GCG CTA CTG AAA GTC (SEQ ID NO:23)          Leu Adapted sequence    255-257 GCG CTG CTG AAA GTC (SEQ IDNO:24)    Leu Unadapted sequence    357-359 AGC CTA GGA CTG TTC (SEQ IDNO:25)    Leu Adapted sequence    357-359 AGC CTG GGA CTG TTC (SEQ IDNO:26)    Leu Human type I collagen 1A2 Sequence (A)    906-908 GCTGGT CCC GCC GGT (SEQ ID NO:27)    Gly Sequence (B)    906-908 GCT GGACCC GCC GGT (SEQ ID NO:28)    GlyDiscussion

In the examples outlined above, RNA was expressed from cDNAs coding forfour different proteins: human and mouse rhodopsin, human peripherin andhuman type I collagen 1A2. Rhodopsin and peripherin have been used toexemplify the invention for retinopathies such as adRP—suppressioneffectors have been targeted to the coding sequences of these genes. Inthe case of the human collagen 1A2 gene a naturally occurringpolymorphism has been used to demonstrate the invention and thepotential use of the invention for disorders such as OI—howevernon-polymorphic regions of the collagen 1A2 gene could be used toachieve suppression. The suppression effectors of choice in theinvention have been hammerhead ribozymes with antisense flanks to definesequence specificity. Hammerhead ribozymes require NUX cleavage sites inopen loop structures of RNA. Notably, other suppression effectors couldbe utilised in the invention and may lead to a more flexible choice oftarget sequences for suppression. Transcripts expressed from all fourgenes have been significantly attacked in vitro using suppressioneffectors directed towards target cleavage sites. In all four examplesthe ribozymes directed to cleavage sites were successful in cleavingtarget RNAs in the predicted manner. Antisense complementary tosequences surrounding the cleavage sites was used successfully to elicitbinding and cleavage of target RNAs in a sequence specific manner.Additionally, transcripts from replacement genes, modified using thedegeneracy of the genetic code so that they code for wild type protein,were protected fully from cleavage by ribozymes.

The utility of an individual ribozyme designed to target an NUX site inan open loop structure of transcripts from a gene will depend in part onthe robust nature of the RNA open loop structure when variousdeleterious mutations are also present in the transcript. To evaluatethis, we analysed RNAPlotFold data for six different adRP causingmutations in the rhodopsin gene. For each of these, the large RNA openloop structure which is targeted by Rz10 was predicted to be maintainedin the mutant transcripts (Table 2A). This is clearly demonstrated inexample 1B (FIG. 4) using a Pro23Leu rhodopsin mutation. Rz10 clearlycleaves the mutant transcript effectively in vitro. The Pro23Leumutation creates a ribozyme cleavage site and can be cleaved in vitro byRz20 a ribozyme specifically targeting this site—however this is not thecase for many mutations. In contrast we have shown that the Rz10ribozyme cleavage site is available for different mutant rhodopsins andcould potentially be used to suppress multiple mutations using asuppression and replacement approach.

In some cases lowering RNA levels may lead to a parallel lowering ofprotein levels however this may not always be the case. In somesituations mechanisms may prevent a significant decrease in proteinlevels despite a substantial decrease in levels of RNA. However in manyinstances suppression at the RNA level has been shown to be effective(see prior art). In some cases it is thought that ribozymes elicitsuppression not only by cleavage of RNA but also by an antisense effectdue to the antisense arms of the ribozyme surrounding the catalyticcore.

In all examples provided ribozymes were designed to cleave at specifictarget sites. Target sites for four of the ribozymes utilised werechosen in open loop structures in the coding regions of transcripts fromthree retinal genes (human and mouse rhodopsin and human peripherin). Inall cases sequence specific cleavage was obtained at the target cleavagesites (FIGS. 1-7). Target sites were chosen in open loop structures tooptimise cleavage. Additionally target sites were chosen such that theycould be obliterated by single nucleotide changes at third base wobblepositions and therefore would code for the same amino acid (Table 2B).In turn this enabled the generation of replacement genes with singlenucleotide alterations which code for wild type protein. In all casestested transcripts from replacement genes were protected from cleavageby ribozymes. Further modifications could be made to replacement genesin wobble positions, for example, to limit the binding ability of theantisense arms flanking the ribozyme catalytic core. The examplesprovided for rhodopsin and peripherin involve suppression of expressionof both disease and wild type alleles of a retinal gene and restorationof the wild type protein using a replacement gene. However, there may besituations where single alleles can be targeted specifically orpartially specifically (PCT/GB97/00574).

In one example, human collagen 1A2, Rz907 was used to target a naturallyoccurring polymorphic site at amino acid 187, (GGA (glycine) -->GGT(glycine), located in an open loop structure from the predicted 2-Dconformations of the transcript (FIG. 9, Table 2B). The ribozyme Rz907cleaved transcripts containing the GGT sequence but transcripts with GGAwere protected from cleavage. Transcripts from both alleles ofindividuals homozygous for the GGT polymorphism could be cleaved byRz907 whereas in the case of heterozygotes cleavage could be directed tosingle alleles (in particular to alleles containing deleteriousmutations PCT/GB97/00574). In both situations replacement genes couldhave the sequence GGA and therefore would be protected from cleavage byRz907. The presence of many such naturally occurring silentpolymorphisms highlights that replacement genes could be modified in asimilar fashion in wobble positions and should produce in most casesfunctional wild type protein. Multiple modifications could be made toreplacement genes at wobble positions which would augment protectionfrom suppression effectors. For example, in situations where antisensenucleic acids were used for suppression, transcripts from replacementgenes with multiple modifications at third base positions would beprotected partially or completely from antisense binding.

In all four examples provided transcripts from cDNA clones were cleavedin vitro in a sequence specific manner at ribozyme cleavage sites.Additionally one base of the ribozyme cleavage site occurs at a wobbleposition and moreover can be altered so as to eliminate the cleavagesite. Ribozyme cleavage sites in the examples given were destroyed bychanging nucleotide sequences so that the consensus sequence forribozyme cleavage sites was broken. However it may be that in some casesthe cleavage site could be destroyed by altering the nucleotide sequencein a manner that alters the 2-D structure of the RNA and destroys theopen loop structure targeted by the ribozyme. cDNAs or DNA fragmentswith altered sequences in the regions targeted by ribozymes weregenerated. RNAs expressed from these cDNAs or DNA fragments wereprotected entirely from cleavage due to the absence of the ribozymecleavage site for each of the ribozymes tested. Of particular interestis the fact that a single nucleotide alteration can obliterate aribozyme target site, thereby preventing RNA cleavage. Althoughribozymes have been used in the demonstration of the invention, othersuppression effectors could be used to achieve gene silencing. Againreplacement genes with altered sequences (at third base wobblepositions) could be generated so that they are protected partially orcompletely from gene silencing and provide the wild type (or beneficial)gene product.

As highlighted before in the text, using the invention the same methodof suppression (targeting coding sequences of a gene) and wherenecessary gene replacement (using a replacement gene with a sequencemodified in third base positions to restore gene expression) may be usedas a therapeutic approach for many different mutations within a givengene. Given the continuing elucidation of the molecular pathogenesis ofdominant and polygenic diseases the number of targets for this inventionis rapidly increasing.

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1. A strategy for suppressing or partially suppressing an endogenousgene and replacing the suppressed gene with a nucleic acid sequence thatdiffers from the endogenous gene, wherein the suppressing agentcomprises at least one suppressor selected from the group consisting ofan antisense nucleic acid, a peptide nucleic acid, a nucleic acidcapable of forming a triple helix, and a ribozyme targeted to theendogenous gene or gene transcript, wherein the replacement nucleic acidsequence encodes at least part of a gene product and is not suppressedby a suppression agent or is suppressed less efficiently by asuppression agent, and wherein the replacement nucleic acid sequencecomprises amino acid codons that encode at least part of the geneproduct, and have modifications at one or more wobble sites such thatreplacement nucleic acid still encodes the wild type or equivalent aminoacids.
 2. A medicament comprising either one or both of a genesuppressing agent and a nucleic acid encoding at least part of areplacement gene product, for use in a strategy as claimed in claim 1.3. A strategy for suppressing or partially suppressing an endogenousgene and introducing a replacement gene the strategy comprising thesteps of: a. providing a suppression nucleic acid able to recognise,bind or cleave an endogenous gene, gene transcript or gene product to besuppressed; and b. providing complete or partial genomic DNA or cDNAencoding a replacement gene, wherein the suppression nucleic acid isunable to recognise, bind or cleave or able to recognise, bind or cleaveless efficiently, equivalent regions in the genomic DNA or cDNA toprevent suppression of the replacement gene, wherein the coding sequenceof the replacement nucleic acid has been altered to prevent or reduceefficiency of suppression and wherein the replacement nucleic acid hasmodifications in one or more wobble sites such that the replacementnucleic acid still codes for the wild type or equivalent amino acids. 4.The use of a strategy as claimed in claim 3 in the preparation of amedicament for the treatment of an autosomal dominant disease caused byan endogenous target gene wherein the disease is caused by differentmutations in the same gene in different patients.
 5. The use of: a. avector containing a suppression effector, the suppression effector ableto recognise, bind or cleave a coding sequence of a target endogenousgene; and b. a vector containing a replacement nucleic acid in the formof genomic DNA, cDNA or RNA, which contains altered wobble sites suchthat the replacement nucleic acid cannot be recognised, bound or cleavedby the suppression effector or are recognised, bound or cleaved lessefficiently by the suppression effector, which suppression effector istargeted towards a coding sequence of the endogenous gene and providesthe wild type gene product and wherein the difference between theendogenous gene and the replacement gene enables the expression of thereplacement gene; in the preparation of a medicament for the treatmentof an autosomal dominant disease caused by the endogenous gene whereinthe disease is caused by different mutations in the same gene indifferent patients.
 6. A use as claimed in claim 5 wherein the diseaseis a polygenic disorder.
 7. A use as claimed in claim 5 wherein thesuppressor and/or replacement gene is administered alone or in a vectorchosen from DNA plasmid vectors, RNA or DNA viral vectors.
 8. A use asclaimed in claim 5 wherein the suppressor and/or replacement gene iscombined with lipids, polymers or other derivatives.
 9. A use as claimedin claim 5 wherein the replacement gene is altered from the wild typegene and provides a beneficial effect when compared to the wild typegene.